POISONING OF HEALTHY HEMATOPOIESIS IS AN UNANTICIPATED MECHANISM DRIVING CLONAL DOMINANCE IN VEXAS SYNDROME [scRNA-seq]

Clonal dominance characterizes hematopoiesis during aging and increases susceptibility to blood cancers and common non-malignant disorders. VEXAS syndrome is a recently discovered adult-onset autoinflammatory disease burdened by a high mortality rate and caused by dominant hematopoietic clones bearing somatic mutations in the UBA1 gene. However, pathogenic mechanisms fueling clonal dominance are unknown. Moreover, the lack of disease models hampers the development of disease-modifying therapies. Here, we performed immunophenotypic dissection of hematopoiesis and single-cell transcriptomics in a VEXAS patient cohort revealing pervasive inflammation across all lineages. Hematopoietic stem/progenitor cells (HSPCs) in VEXAS patients proliferate less, are mostly committed toward myelopoiesis, and prone to egress from the bone marrow. Humanized xenograft models of VEXAS syndrome, generated by inserting the causative mutation in healthy HSPCs through base editing, fully recapitulated patients' hematologic and inflammatory hallmarks. Competitive transplants of VEXAS-like and normal HSPCs showed that while mutant cells are resilient to the inflammatory milieu, wild-type ones are poisoned and progressively overwhelmed by VEXAS clones, becoming unable to support functional multilineage hematopoiesis. Our study unveils an unanticipated mechanism of clonal dominance, provides new models for preclinical investigation of therapeutic strategies, and has relevant implications for clinical management of VEXAS patients. Overall design: scRNA-seq libraries were sequenced on an Illumina NovaSeq6000 with the standard sequencing protocol of R1 28; I1 10; I2 10; R2 90 nt read length. The dataset is composed by 14 samples: 2 samples from human BM cells from UBA1mut and UBA1wt mice harvested 14 weeks post transplantation; 2 samples from sorted human CD34+ HSPCs from the BM of UBA1mut and UBA1wt hematochimeric mice harvested at 14 weeks; 2 samples from murine lineage-negative (Lin-) cells from hematochimeric mice reconstituted by UBA1mut or UBA1wt human cells; 6 samples from BM mononuclear cells, enriched in the CD34+ HSPC fraction, from six patients (PT1, PT2, PT3, PT5, PT8, PT9); 1 samples from human BM cells (enriched for CD34+ HSPC) from VEXAS mice harvested 6 weeks post transplantation; 1 samples from human BM cells (enriched for CD34+ HSPC) from WT mice harvested 6 weeks post transplantation. Both sample are composed of female and male cells. Specifically mice were transplanted with female UBA1wt HSPCs in competition with either male UBA1mut or male UBA1wt HSPCs to distinguish mutant and wildtype cells. Thus, each sample is devided in two during the analysis by probing for XIST gene expression.